T21/DP107, A Synthetic Leucine Zipper-Like Domain of the HIV-1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors This information is current as of October 1, 2021. Shao Bo Su, Ji-liang Gao, Wang-hua Gong, Nancy M. Dunlop, Philip M. Murphy, Joost J. Oppenheim and Ji Ming Wang J Immunol 1999; 162:5924-5930; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. T21/DP107, A Synthetic Leucine Zipper-Like Domain of the HIV-1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors1,2

Shao Bo Su,* Ji-liang Gao,‡ Wang-hua Gong,† Nancy M. Dunlop,* Philip M. Murphy,‡ Joost J. Oppenheim,* and Ji Ming Wang3*

A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant

of migration and calcium mobilization in human and neutrophils. The activity of T21/DP107 on phagocytes was Downloaded from pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected

human embryonic kidney 293 cells. In contrast, fMLP did not induce significant of the same cells at a concentration http://www.jimmunol.org/ as high as 50 ␮M. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca2؉ mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes. The Journal of Immunology, 1999, 162: 5924–5930.

he envelope proteins of HIV-1 are synthesized in the form terminus has a leucine zipper-like motif, whereas another segment, of a precursor, gp160, which is subsequently cleaved by T20/DP178, is located in the carboxyl terminus of the gp41 proteinases to yield mature proteins gp120 and gp41 (1). ectodomain. In the absence of gp120 and the N-terminal fusion T by guest on October 1, 2021 gp120 is noncovalently bound to the extracellular domain of gp41 domain, the ectodomain of gp41 forms a soluble ␣-helical rod-like and mediates viral binding to host cells through high-affinity in- oligomer (4, 5). Synthetic analogues of both T21/DP178 and T20/ teraction with CD4 receptors, followed by interaction with che- DP178 have been shown to inhibit virus-mediated cell- mokine receptors that have recently been identified as HIV-1 fu- and to reduce the infectious titer of cell-free virus (5–11). The sion cofactors (2, 3). The viral envelope gp41 plays a critical role anti-HIV-1 activity of these peptides is presumably due to their in fusion of HIV-1 and host cell membranes (2, 3). Structural anal- competitive association with the native segments on gp41, thus ysis predicts the gp41 ectodomain to contain two segments as ex- blocking the conversion to the fusogenic configuration of the virus. tended helices (4). One segment termed T21/DP107 in the NH2 In the course of investigating the basis for HIV-1-associated suppression of functions, we found that preexposure of human monocytes to either HIV-1 envelope proteins gp120 or

*Laboratory of Molecular Immunoregulation, Division of Basic Sciences; †Intramural gp41 inhibited their chemotactic responses to a wide variety of Research Support Program, Science Applications International Corporation-Freder- chemoattractants, including the bacterial chemotactic peptide ick, National Cancer Institute-Frederick Cancer Research and Development Center, fMLP and a number of recently defined chemokines, through a Frederick, MD 21702; and ‡Laboratory of Host Defenses, National Institute of Al- lergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 mechanism resembling heterologous “desensitization” (12, 13). Received for publication December 10, 1998. Accepted for publication February The inactivation of monocyte chemotactic responses by HIV-1 en- 17, 1999. velope proteins may be responsible for the reduced migratory re- The costs of publication of this article were defrayed in part by the payment of page sponse of monocytes from AIDS patients to a variety of chemoat- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tractants in vitro (14). To further define the structural basis for the 1 S.B.S. is supported in part by a fellowship from the Office of the International capacity of HIV-1 envelope proteins to “desensitize” host cells, we Affairs, National Cancer Institute, National Institutes of Health. evaluated the effects of selected peptide segments of gp41 on hu- 2 The content of this publication does not necessarily reflect the views or policies of man immune cells. We initially found that synthetic C-terminal the Department of Health and Human Services, nor does mention of trade names, peptide segment, T20/DP178, was a potent agonist of FPR, a sev- commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a en-transmembrane, G-protein-coupled receptor on human phago- nonexclusive, royalty-free license in and to any copyright covering the article. cytic cells used by chemotactic N-formyl peptides (Refs. 15 and 3 Address correspondence and reprint requests to Ji Ming Wang, Laboratory of Mo- 16, and Su et al., manuscript in preparation). This led us to further lecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room investigate the effect of synthetic T21/DP107. Here, we report that 31-19, Frederick, MD 21702-1201. E-mail address: [email protected] T21/DP107 activates human monocytes and neutrophils by using

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 5925 two N-formyl peptide receptors (FPR4), the prototype FPR and its lation at sites of inflammation or injury. As shown in Fig. 1, A and variant FPR-like 1 (FPRL1), and exhibits much higher efficacy on B, human PBMC and neutrophils migrated in a dose-dependent FPRL1. manner in response to a concentration gradient of T21/DP107. The chemotactic activity of T21/DP107 was already significant at nM Materials and Methods concentrations for both monocytes and neutrophils, and the cell Reagents and cells response remained high with only slight reduction when T21/ Ϫ5 Ϫ4 The T21/DP107 was synthesized and purified by the Department of Bio- DP107 was used at 10 -10 M (Fig. 1B). In contrast to phago- ϩ chemistry, Colorado State University (Fort Collins, CO), according to the cytes, human CD3 T lymphocytes showed a marginally signifi- published sequence (aa 558–595 of gp41) (5): Ac-NNLLRAIEAQ cant migration (CI ϭ 2) in response to high concentrations (5 ϫ QHLLQLTVWGIKQLQARILAVERYLKDQ-NH2. The purity was 90% or 10Ϫ6 M and higher, Fig. 1B), indicating the effect of T21/DP107 more, and the amino acid composition was verified by mass-spectrometer. The endotoxin levels in dissolved peptide were undetectable. The synthetic is mainly on phagocytic cells. We next examined whether phago- formyl peptide fMLP was purchased from Sigma (St. Louis, MO). The cyte migration induced by T21/DP107 was dependent upon the human PBMC were isolated from leukopacks through the courtesy of effect of chemotaxis or chemokinesis. Checkerboard analyses Transfusion Medicine Department (National Institute of Health Clinical showed that monocytes migrated when higher concentrations of Center, Bethesda, MD). Monocytes were further purified by ellutriation to yield Ͼ90% purity. Human neutrophils were purified from the same leu- T21/DP107 were present in the lower wells of the chemotaxis kopacks by 3% dextran sedimentation with a purity of Ͼ98%. Rat baso- chamber (Table I). There was no enhanced cell migration when philic leukemia cells stably transfected with epitope-tagged FPR (ETFR) higher concentrations of T21/DP107 were present in the upper were a kind gift of Drs. H. Ali and R. Snyderman (Duke University, Durham, NC). The cells were designated ETFR and were grown in wells. With equal concentrations of T21/DP107 in both upper and DMEM, 10% FCS, and 0.8 mg/ml geneticin (G418) to maintain selection lower wells, a slightly increased monocyte migration was ob- Downloaded from pressure. The FPRL1 cDNA was cloned and transfected into human em- served. These results suggest that the cell migration induced by bryonic kidney cells 293 (designated FPRL1/293 cells), as reported previ- T21/DP107 was due mainly to a chemotactic effect with minor ously (17). The cells were maintained in DMEM, 10% FCS, and 2 mg/ml geneticin (G418). contribution of chemokinesis. The migration of monocytes and neutrophils to T21/DP107 was completely inhibited by pretreat- Chemotaxis ment of the cells with pertussis toxin, but not by cholera toxin or

Leukocyte, ETFR, and FPRL1/293 cell migration was assessed using a herbimycin A (Fig. 1C, and data not shown), suggesting that a http://www.jimmunol.org/ 48-well microchemotaxis chamber technique, as previously described (18– G-protein of the Gi type-coupled receptor was involved (15, 16, 20). Different concentrations of stimulants were placed in wells of the 21–24). This was supported by the potent induction of a dose- lower compartment of the chamber (Neuro Probe, Cabin John, MA), and 2ϩ the cell suspension was seeded in wells of the upper compartment, which dependent, and pertussis toxin-sensitive, calcium (Ca ) mobili- was separated from the lower compartment by a polycarbonate filter (Os- zation in monocytes and neutrophils by T21/DP107 (Fig. 2, A and monics, Livermore, CA; 5-␮m pore size for leukocytes, 10-␮m pore size C). The Ca2ϩ-mobilizing activity of T21/DP107 was also signif- ϩ for ETFR and FPRL1/293 cells). For CD3 T lymphocytes, the filters were icant at nM concentrations, indicating that this synthetic peptide is precoated with 20 ␮g/ml bovine fibronectin (Sigma). The filters for ETFR and FPRL1/293 cell migration were precoated with 50 ␮g/ml collagen type a potent activator of human phagocytic cells. The possibility that byproduct(s) formed during peptide synthesis/purification might

I (Collaborative Biomedical Products, Bedford, MA) to favor the attach- by guest on October 1, 2021 ment of the cells. After incubation at 37°C (90 min for monocytes, 60 min account for the activity of T21/DP107 was unlikely, since a fusion for neutrophils, 180 min for T cells, and 300 min for ETFR or FPRL1/293 peptide (aa 517–532 of gp41) synthesized during the same period cells), the filters were removed, stained, and the cells migrating across the 2ϩ filter were counted by light microscopy after coding the samples. The ex- as T21/DP107 did not posses any chemotactic or Ca -mobilizing periments were performed at least five times with each cell type, and the activity in phagocytes (data not shown). results are presented as the chemotaxis indexes (CI) representing the fold To characterize the molecular nature of the receptor(s) possibly increase in the number of migrating cells in response to stimuli, over the used by T21/DP107 on phagocytic cells, a series of cross-desen- spontaneous cell migration (in response to control medium). The signifi- cance of the increase in cell migration was determined using Student’s t sitization experiments were performed by using a variety of che- ϩ test, and CI Ն 2 was statistically significant compared with medium control moattractants. T21/DP107 did not desensitize the Ca2 flux in (at least p Ͻ 0.05). monocytes or neutrophils induced by chemokines, such as mono- Calcium mobilization cyte chemoattractant protein (MCP)-1, RANTES, MCP-3, mac- rophage-inflammatory protein-1␣, IL-8, and stromal cell-derived 7 Calcium mobilization was assayed by incubating 10 cells/ml of mono- factor-1␣ (data not shown). Therefore, T21/DP107 does not share cytes, neutrophils, FPRL1, or ETFR transfectants in loading buffer con- a receptor with any of these chemokines. However, high concen- taining 138 mM NaCl, 6 mM KCl, 1 mM CaCl2, 10 mM HEPES (pH 7.4), 5 mM glucose, and 0.1% BSA with 5 ␮M fura-2 (Sigma) at 37°C for 30 trations (Ն1 ␮M) of the bacterial chemotactic N-formylated pep- min. The dye-loaded cells were washed and resuspended in fresh loading 6 tide fMLP had a partial desensitizing effect on T21/DP107-induced buffer. The cells were then transferred into quartz cuvettes (10 cells in 2 2ϩ ml), which were placed in a luminescence spectrometer LS50 B (Perkin- Ca mobilization in both monocytes and neutrophils (Fig. 2, B Elmer Limited, Beaconsfield, U.K.). Stimulants at different concentrations and D). In contrast, T21/DP107 did not significantly desensitize were added in a volume of 20 ␮l to the cuvettes at indicated time points. the effect of fMLP (Fig. 2, B and D). These results suggest that The ratio of fluorescence at 340- and 380-nm wavelength was calculated T21/DP107 may share receptor(s) with fMLP on human phago- using the FL WinLab program (Perkin-Elmer). Unless specified, all experiments were performed at least five times with cytic cells and may have preference on a receptor for which fMLP similar results, and data shown in this study were from representative has lower affinity. experiments. Since fMLP was known to induce Ca2ϩ in phagocytes through at least two seven-transmembrane, G-protein-coupled receptors, Results FPR and FPRL1 (15–17), we tested the effect of T21/DP107 on We first tested whether synthetic T21/DP107 could induce human these two receptors transfected and overexpressed in human cells leukocyte migration, a crucial step for cell homing and accumu- that originally were not responsive to fMLP stimulation. fMLP over a wide range of concentrations induced Ca2ϩ mobilization in

4 Abbreviations used in this paper: FPR, formyl peptide receptor; FPRL1, FPR-like 1; FPR-transfected rat basophil leukemia cell line (ETFR cells), with ETFR, epitope-tagged FPR; CI, chemotaxis index; SAA, serum amyloid A. minimal effective dose at low pM concentration range (Fig. 3A). In 5926 ACTIVATION OF FPR/FPRL1 BY T21/DP107

FIGURE 1. Induction of phagocyte mi- gration by T21/DP107. Different concentra- tions of T21/DP107 were placed in the lower wells of the chemotaxis chamber; cell sus- pension was placed in the upper wells. The upper and lower wells were separated by polycarbonate filters. After incubation, the Downloaded from cells migrated across the filters were stained and counted. A, Visualization of monocyte and neutrophil migration in response to T21/ DP107 (1 ␮M) and fMLP (100 nM). B, Fold increase of leukocyte migration in response to T21/DP107 over control medium. C, In- hibition of monocyte migration in response http://www.jimmunol.org/ to T21/DP107 by pretreatment of the cells with pertussis toxin (PT; 100 ng/ml, 30 min at 37°C). by guest on October 1, 2021

contrast, the minimal effective concentration for fMLP to induce vious observation that FPR is a high-affinity receptor for fMLP, Ca2ϩ mobilization in FPRL1-transfected cells (FPRL1/293 cells) whereas FPRL1 has much lower affinity (15–17). The synthetic was at low ␮M range (Fig. 3D). These results confirmed the pre- T21/DP107 also induced Ca2ϩ mobilization in cells transfected

Table I. Checkerboard analysis of monocyte migration in response to T21/DP107a

Number of Migrated Cells in 1HPF (Mean Ϯ SE)

T21 in T21 in upper wells (M) Lower Wells (M) Medium 10Ϫ8 10Ϫ7 10Ϫ6

Medium 20 Ϯ 111Ϯ 27Ϯ 19Ϯ 2 10Ϫ8 40 Ϯ 4b 28 Ϯ 37Ϯ 210Ϯ 3 10Ϫ7 64 Ϯ 3b 43 Ϯ 2b 25 Ϯ 426Ϯ 3 10Ϫ6 132 Ϯ 6b 110 Ϯ 3b 89 Ϯ 6b 57 Ϯ 4b

a Different concentrations of T21/DP107 were placed in the upper and/or lower wells of the chemotaxis chamber; monocytes at 2 ϫ 106/ml were placed in the upper wells. The upper and lower wells were separated by a polycarbonate filter. After incubation, the nonmigrating cells were removed, and the filter was fixed, stained, and the cells migrated across the filter were counted in three high-powered fields (HPF; 400ϫ). The results are expressed as the mean value (ϮSE) of the cells in 1 HPF. Similar results were obtained in two separate experiments. b A value of p Ͻ 0.01 compared with migration in the presence of medium alone in both upper and lower wells, as determined by Student’s t test. The Journal of Immunology 5927 Downloaded from

FIGURE 2. Calcium (Ca2ϩ) mobilization in phagocytes induced by T21/DP107. Human monocytes (A) or neutrophils (C) were loaded with fura-2 and then were stimulated with various concentrations of T21/DP107. The ratio of fluorescence at 340 and 380 nm wave length was recorded and calculated ␮ 2ϩ ␮ using the FL WinLab program. B and D, Desensitization of T21/DP107 (1 M) induced Ca flux by fMLP (1 M) in monocytes (B) or neutrophils (D). http://www.jimmunol.org/ with either of these receptors (Fig. 3, B and E). However, the desensitization of Ca2ϩ flux between T21/DP107 and fMLP in minimal effective dose for T21/DP107 to activate FPRL1 was at both receptor transfectants. As shown in Fig. 3, C and F, although nM range as compared with low ␮M range on FPR, suggesting that sequential stimulation of the cells expressing FPR or FPRL1 with T21/DP107 activates FPRL1 with higher efficacy. A comparison T21/DP107 and fMLP resulted in bidirectional desensitization, a for the efficacy between T21/DP107 and fMLP on two receptors 1000-fold excess of fMLP was required to desensitize the effect of could be better illustrated by the requirement of the concentrations T21/DP107 in FPRL1/293 cells. Conversely, a much higher con- to elicit an equal response by these two agents. In FPR-expressing centration of T21/DP107 was necessary to completely abolish the by guest on October 1, 2021 cells, to induce a change in the ratio of 2 at 340/380 nm wave- subsequent response to fMLP of the cells transfected with FPR. In length fluorescence, 10Ϫ10 fMLP vs 10Ϫ5 T21/DP107 were re- control experiments, T21/DP107 and fMLP did not induce any quired; whereas in FPRL1/293 cells, 5 ϫ 10Ϫ5 fMLP vs 5 ϫ 10Ϫ7 Ca2ϩ mobilization in parental or mock-transfected rat basophil cell T21/DP107 induced a change in the ratio of 0.6. Such comparison line and human embryonic kidney 293 cells (data not shown). indeed indicates T21 to be a more potent agonist for FPRL1 com- These results further support the notion that FPR and its variant, pared with fMLP. This was further supported by results of cross- FPRL1, are differentially activated by fMLP and T21/DP107.

FIGURE 3. Calcium mobilization in ETFR and FPRL1/293 cells induced by T21/DP107. The FPR-transfected rat basophil cell line (RBL), ETFR cells (A and B), and FPRL1/293 transfectants (D and E) were used to evaluate Ca2ϩ flux induced by T21/DP107 or fMLP. C and F, Cross-desensitization of cell signaling between T21/DP107 and fMLP. 5928 ACTIVATION OF FPR/FPRL1 BY T21/DP107

FIGURE 4. Migration of ETFR cells and FPRL1/ 293 cells in response to T21/DP107. Different concen- trations of T21/DP107 were placed in the lower wells of the chemotaxis chamber; cell suspension was placed in the upper wells. The upper and lower wells were separated by polycarbonate filters precoated with mouse collagen type I. After incubation, the cells migrated across the filters were stained and counted (A). CI represents the fold increase of leukocyte mi-

gration in response to T21/DP107 over control me- Downloaded from dium. B, Migration of FPRL1/293 cells. C, Migration of ETFR cells. http://www.jimmunol.org/

The chemotactic response of the cells transfected with FPR or supernatants, providing evidence in support of them being biolog- by guest on October 1, 2021 FPRL1 was tested as another sensitive parameter to assess the ically relevant ligands for FPR on phagocytic cells. Mitochondrial receptor targets of T21/DP107 (18–20). FPRL1/293 cells showed proteins are also N-formylated and are chemotactic for neutrophils a marked migratory response to T21/DP107 with an EC50 of 50 (26), thus constituting an endogenous source of chemotactic pep- nM (Fig. 4, A and B), but these cells failed to migrate in response tides released by damaged tissue. Although early studies indicated to a wide range of concentrations of fMLP (Fig. 4B). In contrast, that the N-formyl group was essential for optimal agonist potency ETFR cells were induced to migrate by fMLP at nM range con- (27), recent studies have shown that nonformylated peptides may centrations, but much higher concentrations of T21/DP178 were also attract and activate phagocytes (15, 16). The prototype recep- required to induce the migration of the same cells (Fig. 4C). The tor for formyl peptides designated FPR is expressed by neutrophils chemotaxis experiments indicate that fMLP is only a partial ago- and monocytes and was cloned several years ago (15, 16). The nist for FPRL1, since it did not induce FPRL1-expressing cells to FPR was subsequently shown to have a much broader spectrum of migrate. T21/DP107, on the other hand, appears to be an efficient agonists than initially expected. In fact, the synthetic pentapeptide agonist on both FPR and FPRL1, although the efficacy for FPR- Met-Nle-Leu-Phe-Phe-OH, either N-formylated or N-acetylated, is expressing cells was much lower than for FPRL1-expressing cells. more potent than the parental prototype fMLP in the induction of Ca2ϩ flux in human neutrophils (24). Amino terminal urea-substi- Discussion tuted and carbonate-modified peptides have also been shown to be In this study, we observed that T21/DP107, a synthetic peptide potent agonists for FPR (28, 29). Structural analysis of FPR sug- domain of the HIV-1 gp41, potently attracts and activates human gests that the binding pocket of this receptor is able to accommo- phagocytic cells by using two seven-transmembrane, G-protein- date an amino-terminal group larger than a formyl group (28, 29), coupled receptors, FPR and FPRL1, with higher efficacy on and this may explain the capacity of this receptor to interact with FPRL1. Although these receptors were identified and cloned sev- a great variety of endogenously derived, as well as exogenous, eral years ago, their precise role in host defense has not been fully ligands. elucidated (15, 16). Leukocyte infiltration at the sites of inflam- FPRL1 was identified and molecularly cloned from human mation in vivo is considered to be based on migration of cells phagocytic cells by low-stringency hybridization of the cDNA li- toward a gradient of chemoattractant(s), either derived from mi- brary with the FPR sequence and was initially defined as an orphan croorganisms or the local tissue. The discovery of synthetic N- receptor (17, 30–32). The cloning of the same receptor, termed formyl oligopeptide chemoattractants for phagocytes represented a FPRH2, from a genomic library was also described (33). FPRL1 major advance in the study of leukocyte locomotion (25). Several possesses 69% identity at the amino acid level to FPR (15, 16), and natural N-formyl peptide chemoattractants, including the prototype both receptors are expressed by monocytes and neutrophils and are N-formyl peptide, fMLP, have since been purified from bacterial clustered on human chromosome 19q13 (33, 34). While fMLP is a The Journal of Immunology 5929 high-affinity agonist for FPR, it interacts with and induces Ca2ϩ cyte migration and activation by its agonists suggest that FPRL1 flux in FPRL1 only at high concentrations (Fig. 3D, and Refs. 17, and FPR may share many signal transduction steps following ac- 31, and 34). In our study, fMLP did not induce significant migra- tivation. The binding of FPR by agonists results in a G-protein- tion of FPRL1/293 cells at a concentration as high as 50 ␮M(5ϫ mediated signaling cascade leading to cell adhesion, chemotaxis, 10Ϫ5 M; Fig. 4B), suggesting that fMLP is not a full agonist for release of oxygen intermediates, enhanced phagocytosis, and bac- FPRL1. In contrast, T21/DP107, although also activating both re- terial killing, as well as mitogen-activated protein kinase activation ceptors, showed a much higher efficacy on FPRL1 and induces leading to gene transcription (15, 16). Activation by fMLP can also migration of FPRL1/293 cells at nM concentrations. Thus, com- lead to heterologous desensitization of the subsequent cell pared with fMLP, T21/DP107 is a functionally more relevant ag- response to other G-protein receptor ligands (46, 47), includ- onist for FPRL1. Although FPRL1 is mainly expressed in mono- ing chemokines. In our previous study, incubation of human cytes and neutrophils, cells other than phagocytes, such as phagocytes with FPRL1 agonist SAA resulted in a reduction of hepatocytes, have also been shown to express FPRL1 (15). Re- cell responses to a number of chemoattractants (38), suggesting cently, the expression of this receptor has been reported to be that activation of FPRL1 may also activate signaling events that highly inducible in epithelial cells by specific , such as cause desensitization of other G-protein-coupled chemotactic IL-13 and IFN-␥ (35). Therefore, FPRL1 may play an important receptors. role in inflammatory and immunological responses in human cells. The relevance of our current findings to the course of disease In support of this notion, we recently identified FPRL1 to be a effects on host and benefits, if any, to HIV-1 infection remains to functional receptor for a normal serum protein, serum amyloid A be established. However, our observations do suggest some spec- (SAA) (36), which increases its concentration by up to several ulation possibilities. It has been reported that monocytes and neu- hundred-fold during acute phase responses and is a potent phago- trophils isolated from HIV-1-infected patients responded poorly to Downloaded from cyte chemoattractant and activator (37, 38). SAA and T21/DP107 a variety of chemoattractants, including fMLP (14, 48–51) in attenuated each other’s Ca2ϩ-mobilizing activity in FPRL1/293 vitro. We have found that recombinant soluble gp41 of the HIV-1 cells, indicating that these two chemoattractants share FPRL1 as is able to potently down-regulate the expression and function of their functional receptor (Su et al., data not shown). It should be fMLP receptor and the receptors for a variety of chemokines on noted that T21/DP107 does not bear any significant sequence ho- monocytes, including CCR5 and CXCR4, two major HIV-1 fusion mology to either fMLP or SAA. Therefore, FPRL1, like its pro- cofactors (13). Intriguingly, the effect of gp41 on monocytes is http://www.jimmunol.org/ totype FPR, is also capable of hosting a broad spectrum of ligands. dependent on the presence of cellular CD4, another fusion receptor It should be pointed out that in our study, human CD3ϩ T lym- of HIV-1 (13). It is not clear whether soluble gp41 itself is capable phocytes showed a weak migration in response to high of interacting with FPR or, alternatively, conjugation with CD4 concentrations of T21/DP107. Whether this low level migration may cause exposure of its domains to interact with these receptors. is also mediated by the presence of FPRL1 in T cells is under Also, further study is needed to examine whether HIV-1 envelope investigation. proteins undergo proteolytic cleavage in vivo to yield peptide frag- In addition to peptide and protein agonists, a lipid metabolite ments that interact with FPR and/or FPRL1. In a parallel study, we lipoxin A4 (LXA4) has been reported to be a high-affinity ligand found that synthetic peptide T20/DP178 of the gp41 C-terminal by guest on October 1, 2021 and potent agonist for FPRL1 (also termed LXA4R) (39). LXA4 is domain was a potent and selective FPR agonist, while its ana- an eicosanoid generated during a number of host reactions, such as logues lacking several amino acids were FPR antagonists (Su et inflammation, thrombosis, and atherosclerosis, and was initially al., manuscript in preparation). This, together with the present ob- discovered as an inhibitor of immune responses (reviewed in Ref. servation of T21/DP107, suggests that gp41 may possess multiple 40). LXA4 was subsequently reported to inhibit neutrophil che- domains that could potentially interact with cellular receptors, thus motaxis (41) and transepithelial migration induced by chemotactic affecting the immune responses. It has been reported that gp41 Ag agents (42). LXA4 bound to Chinese hamster ovary cells trans- could be detected in brain tissues of AIDS dementia (52), and Abs fected with FPRL1(LXA4R) with high affinity and increased recognizing various epitopes of gp41 appear at early stages of GTPase activity and the release of esterified arachidonate (39). HIV-1 infection (53). In fact, we found that both synthetic T21/ Thus, LXA4 has been proposed to be an endogenously produced DP107 and T20/DP178 epitopes of gp41 were recognized by sera ligand for FPRL1 (39, 43). Although LXA4 has not been docu- from AIDS patients by immunoblotting (data not shown), suggest- mented to induce Ca2ϩ mobilization in neutrophils or FPRL1- ing that gp41 and its epitopes are accessible to host cells, including transfected cells (39), it was reported to induce Ca2ϩ flux and APCs. Therefore, although the receptors for formylated peptides chemotaxis in monocytes, presumably through FPRL1 (44, 45). such as FPR and FPRL1 are not used by HIV-1 for fusion, they Based on these observations, differential activation of second mes- may participate in the regulation of host innate immune responses sengers in monocytes vs neutrophils by LXA4 was postulated. In seen in AIDS patients characterized by an initial stimulation of our study, we did not detect significant induction of Ca2ϩ flux or immune system in the early stage of the disease followed by pro- chemotaxis in FPRL1/293 cells by a commercially available gressive immunosuppression. LXA4 (Biomol, Plymouth Meeting, PA), nor did we observe in- hibition of T21/DP107 signaling by this LXA4 in either phago- Acknowledgments cytes or FPRL1/293 cells. Since LXA4 is a highly unstable lipid We thank Drs. H. Ali and R. 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